Neuronal calcium channels are well-known targets for inhibition by receptor-G protein pathways, and multiple forms of inhibition have been described. Transmitter-mediated inhibition of neuronal calcium current is a transient phenomenon. Little is known about the mechanisms of desensitization of transmitter-mediated modulation of calcium channels. Data from our laboratory have shown that two families of proteins are involved in the process of desensitization of transmitter-mediated inhibition of N type calcium current. The onset of desensitization requires the activation of an endogenous G protein coupled receptor kinase called GRK3. Regulators of G protein signaling, RGS proteins, determine the rate of desensitization by accelerating the rate of GTP hydrolysis. Our results show that RGS proteins selectively accelerate the rate of desensitization of G/i and G/o-mediated pathways. We have described a set of protein interactions at the level of the Ca2+ channel by which endogenous RGS 12 selectively increases the duration of the GABA/B-activated, G/o-, tyrosine kinase-mediated inhibition of calcium current. How the regulation of receptor function by GRKs and regulation of G protein function by RGS proteins cooperate in the overall desensitization process is not understood and form the focus of studies in this term of the grant. Experiments described in this proposal will employ a combination of biochemical, molecular and electrophysiological approaches to study at the single-cell level the interactions between the receptors and G proteins involved in the mechanisms of desensitization. Aim 1 will determine whether differences in the alpha 2-adrenergic receptor domains targeted for GRK phosphorylation give rise to different time courses of inhibition of calcium current including the desensitization phase. Aim 2 experiments will determine how the activation of GRK3 triggers the onset of desensitization. Aim 3 will evaluate the importance of interactions between the receptor kinase GRK3 and Gbetagamma isoforms for desensitization. Aim 4 analyzes the role of the domains of RGS proteins in the desensitization process. For this, chimeric RGS proteins will be used to determine the molecular domains in RGS proteins that confer selectivity between G/o-mediated pathways. Understanding these mechanisms will pave the way for the development of new drugs useful in the treatment of disorders associated with elevated neurotransmitter levels, such as stroke, or clinical problems, such as drug tolerance, that are associated with unnaturally high extracellular hormone or drug concentrations.